Evaluation of the viability of paeonol against human Demodex
The mites were observed under a light microscope (Nikon, Tokyo, Japan). The initial state of the mites revealed that their holonomic bodies were active, with five or six tarsi; they also moved 6–8 times in a minute (Fig. 2A and D). Following exposure to the paeonol solution, the bodies of both D. folliculorum and D. brevis significantly contracted and twisted; the body condition (more than six tarsi moved for more than 10 times in 1 minute) was more strenuous than the initial activity state (Fig. 2B and E). After considerable struggle, their bodies gradually returned to their original appearance. The movement of the mites was delayed, podosoma and gnathosoma were expanded, opisthosoma was shortened, and the bodies of the mites became slightly more transparent than their primal form on death. There was no sign of movement in their pincers and tarsi for 1 min, even after these parts were stimulated with a skinny anatomical needle, particularly in the 60, 50, and 40 mg/mL concentration groups (Fig. 2C and F). In the final stage of the 8-hour observation period, the mites showed weak motor performance; only one leg moved 1–2 times in 1 min in response to the abovementioned stimulation, and the bodies of the mites did not change in the 30 mg/mL concentration group. The groups treated with paeonol at 10 or 20 mg/mL concentrations showed relatively vigorous movements without any needle, with the movement of two or three tarsi 3–5 times per minute.
Evaluation of the acaricidal effect of paeonol on D. folliculorum
As shown in Table 1, no recorded deaths occurred in the pure paraffin oil negative control group. However, in the test groups, mite death was observed in the 60, 50, 40, 30, 20, and 10 mg/mL paeonol solutions containing paraffin oil for 8 h. Hence, the average dead time of killing mites at 60, 50, and 40 mg/mL paeonol concentrations was 118, 142.2, and 240 min, respectively; furthermore, the average dead time was more than 480 min for 30, 20, and 10 mg/mL paeonol concentrations. The mortality rates in 8 h were 100%, 100%, 90.3%, 6.0%, 3.1%, and 3.3%, respectively. The results showed that at decreasing drug concentrations, the acaricidal effect faded over time, and the death time increased incrementally. No significant difference was observed between the 30 mg/mL group and the negative control group, the 20 mg/mL group and the negative control group, and the 10 mg/mL group and the negative control group (χ2 = 0.001, P > 0.05; χ2 = 0.001, P > 0.05; χ2 = 0.001, P > 0.05) based on the statistical analysis of the 8-h mortality period. A significant difference in mortality was noted between the 60, 50, and 40 mg/mL groups and the negative control group (χ2 = 34.844, P < 0.05; χ2 = 35.757, P < 0.05; χ2 = 24.468, P < 0.05). Therefore, the 40 mg/mL dose was the lowest effective concentration of paeonol on D. folliculorum in this study.
Evaluation of the acaricidal effect of paeonol on D. brevis
Table 2 shows that t the average dead time was 98.6, 123.4, and 181 min for the 60, 50, and 40 mg/mL paeonol concentrations, respectively. Additionally, more than 480 min was required to kill mites at 30, 20, and 10 mg/mL concentrations. In the negative controls, all mites remained active. The mortality rates in 8 h were 100%, 100%, 92.6%, 17.8%, 7.1%, and 7.1% for 60, 50, 40, 30, 20 and 10 mg/mL concentrations, respectively. No apparent difference in the mortality rate was noted between the 30, 20, and 10 mg/mL groups and the negative control group (χ2 = 1.239, P > 0.05; χ2 = 0.440, P > 0.05; χ2 = 0.440, P > 0.05, respectively). However, the 60, 50, and 40 mg/mL groups showed a significant difference when compared with the negative control group (χ2 = 35.571, P < 0.05; χ2 = 36.512, P < 0.05; χ2 = 28.270, P < 0.05, respectively). Thus, the 40 mg/mL dose was the lowest effective concentration of paeonol against D. brevis.
The acaricidal effect of paeonol on the two species of human Demodex
Tables 1 and 2 and Fig. 3 suggest that the average dead time for D. brevis (98.6, 123.4, and 181 mins) was less than that of D. folliculorum (118.2, 142.2, and 240 mins) in 60, 50, and 40 mg/mL concentration groups, respectively. Thus, an apparent difference was noted between D. brevis and D. folliculorum in the average dead time at the same concentration groups. D. folliculorum (81, 72, 145, 365, 422, and 436 mins) showed a longer survival time than D. brevis (59, 63, 96, 322, 391, and 391) at the same concentration groups of paeonol. The death rates of D. folliculorum in 8 h were 100%, 100%, 90.3%, 6.0%, 3.1%, and 3.3% for 60, 50, 40, 30, 20, and 10 mg/mL paeonol concentrations, respectively; the death rates of D. brevis were 100%, 100%, 92.6%, 17.8%, 7.1%, and 7.1% for 60, 50, 40, 30, 20, and 10 mg/mL paeonol concentrations, respectively. Additionally, paeonol could kill more D. brevis than D. folliculorum, though higher drug concentrations resulted in faster death for both mite species. The average dead time decreased with the increasing concentration of paeonol for both mite species. Thus, paeonol exhibited superior acaricidal activity against D. brevis than against D. folliculorum in terms of average dead time, survival time, and mortality rate in 8 h.